Physical and chemical properties of Coarse Woody Debris submitted to the natural process of decomposition in a Secondary Atlantic Forest Fragment in Brazil

Coarse Woody Debris (CWDs) are constantly exposed to the natural decomposition process of wood, which can lead to a change in its physical–chemical properties. However, these changes have not yet been fully elucidated, requiring further studies to help to understand the effect of this process on CWDs degradation. Thus, the objectives of this study were: (i) verify if the decomposition affects the physical–chemical properties of the CWDs; (ii) verify if the structural chemical composition of the CWDs is altered as a function of decomposition, using immediate chemical and thermogravimetric analysis. Wood samples were collected from the CWDs to carry out these analyses, considering pieces with diameters ≥ 5 cm separated into 4 decay classes. The results indicated that the average apparent density decreased as a function of the increase of CWDs decomposition (0.62–0.37 g cm−3). The averages contents of Carbon and Nitrogen suffered less impact with the increase of CWDs decompositions, ranging from 49.66 to 48.80% and 0.52 to 0.58%, respectively. Immediate chemical and thermogravimetric analysis indicated a loss of holocelluloses and extractives and an increase in the concentration of lignin and ash throughout the decomposition process. The weight loss analyzed by thermogravimetric analysis was greater for less decomposed CWDs and with larger diameters. The use of these analyzes removes the subjectivity of CWDs decay classes, reducing the number of tests to determine CWDs physical–chemical properties and increasing the studies accuracy focused on the carbon cycle of these materials.

www.nature.com/scientificreports/ CHN/CHNS/O; St. Joseph, MI). In this method, the gases emitted by samples burning at 1050ºC were quantified by an infrared detector, which allows the determination of the content, in %, of these elements 39 . The C/N ratio of CWDs was calculated as an indicator of the natural decomposition of these materials on the forest floor.
Immediate chemical analysis. Volatile materials (Vol), ash content (Ash) and fixed carbon (FC) were quantified using a Linn Elektro Therm® muffle furnace, according to the ASTM standards D1762-84 53 . The calculations to determine these parameters, in %, were based on the following expressions: www.nature.com/scientificreports/ where P is the original mass of the sample, in g; P 0 is the original mass of the crucible, in g; P 1 is the initial mass of the crucible + the mass of the sample, in g; P 2 is the final mass of the crucible + the mass of the sample, in g 54,55 .
Thermogravimetric analysis (TG/DTG). The thermogravimetric analysis of CWDs was performed using a DTG-60H Shimadzu equipment, under a nitrogen atmosphere, with a constant flow rate of 50 ml min −1 . Thermograms were obtained from a temperature of 100 °C to a temperature of 450 °C, with a heating rate of 10 °C min −1 . The thermogravimetric curves (TG) of CWDs were analyzed by diameter and decay classes to evaluate the weight loss as a function of temperature, while the first derivative of the thermogravimetric curve (DTG) was obtained to identify the temperatures at which the highest weight losses occurred. Weight losses were calculated from TG curve for the following temperature ranges: 100-200 °C; 200-300 °C; 300-450 °C. The residual weight was obtained at a temperature of 450 °C, considering the mass of the absolutely dry sample at a temperature of 100 °C as the initial value.
Statistical analysis. Analysis of Variance (ANOVA) and Tukey's post-hoc test was applied to test whether the means of the physical (apparent density) and chemical (elementary and immediate chemical analysis) parameters of the CWDs differed statistically between the decay classes. The Shapiro-Wilk test was performed to test the assumption of normality in the following thermogravimetric analysis datasets: (i) weight losses by decay classes of CWDs, in 3 temperature ranges; (ii) residual weight by decay classes of CWDs; (iii) residual weight by diameter classes of CWDs. Only the dataset with losses weight at temperatures of 100-200 °C and 200-300 °C violated the assumption of data normality (P < 0.05). In these cases, the nonparametric Kruskal-Wallis test was applied to test whether there were statistical differences between the medians of the evaluated groups. In the other data sets in which normality was reached (P > 0.05), Analysis of Variance (ANOVA), followed by Tukey's post-hoc test, were applied to test whether there were statistical differences between the means of the evaluated groups. Spearman's correlation was calculated to quantify the association degree of two nonparametric variables in intensity and direction 16,56,57 . The correlation matrix was generated by the software R Core Team 58 using the thermogravimetric, physical and chemical properties of CWDs.

Results
Physical properties. Apparent density. The apparent density (mean ± standard deviation) decreased as a function of the decay class of CWDs, ranging from 0.62 g cm −3 (± 0.13 g cm −3 ) for the least decomposed residue (class 1) to 0.37 g cm −3 (± 0.17 g cm −3 ) for the most decomposed (class 4). The apparent average densities did not follow a pattern of increase or decrease in value as a function on the CWD's diameter classes variation (Table 2).

Chemical properties. Elementary chemical analysis. Carbon (C) contents (mean ± standard deviation)
showed a low variation between the CWD decay classes: 49.66% (± 0.90) to 48.80% (± 1.39%). This same behavior was observed for Nitrogen (N) contents (mean ± standard deviation), which ranged from 0.44% (± 0.08%) to 0.58% (± 0.20%). The C/N ratio ranged from 88.14 (± 19.86) to 116.61 (± 21.72), with the lowest values for the highest decomposition classes. The CWD's diameter classes did not significantly affect these parameters and they did not show a well-defined behavior pattern (Table 3).  Table 3. Carbon Contents-C (%) (mean ± standard deviation), Nitrogen-N (%) (mean ± standard deviation) and C/N Ratio (mean ± standard deviation) by diameter and decay classes of CWDs. Decay classes: (i) Materials that have just fallen to the ground with leaves and bark intact; (ii) Materials similar to those of class "i", but with the bark showing rotting or peeling; (iii) Materials with a high stage of decomposition and showing some resistance to being broken; (iv) Materials that are rotten, friable and without resistance to being broken. Means followed by the same letter do not differ statistically by Tukey's test, at the 5% significance level.

Diameter Classes (cm)
Decay classes www.nature.com/scientificreports/ Immediate chemical analysis. The volatile materials (mean ± standard deviation) contained in the CWDs ranged from 67.71% (± 5.70%) to 81.59% (± 2.63%) while the ash content (mean ± standard deviation) showed values ranging from 1.47% (± 0.36%) to 10.07% (± 5.89). The CWDs Fixed Carbon contents ranged from 16.63% to 22.22%. The parameters of the immediate chemical analysis did not show a pattern of behavior as a function of the increase in the CWD's diameter classes (Table 4).
Thermogravimetric analysis (TG/DTG). The wood degradation components occurred in a narrow temperature range, partially overlapping, where: (i) water loss (0-100 °C); (ii) degradation of hemicellulose (225-275 °C); (iii) cellulose degradation (275-375 °C); and (iv) lignin degradation (> 370 °C). Thermogravimetric curves (TG/DTG) were obtained for each CWD diameter and decay class (Fig. 2). The curves indicated that thermal degradation profiles of CWDs suffered variations in the residual masses and in the maximum peaks of woods constituent's degradations. Thermogravimetric curves (TG/DTG) indicated that thermal degradation profiles suffered variations in the residual masses and in the maximum peaks of woods constituent's degradations according to the diametric and decomposition class of the CWDs. Weight losses for the first decay class (G1) were similar. However, a longer length of the DTG curve (close to 287 °C) was observed for the larger diameter sample. The second DTG peak (close to 351 °C) showed greater weight loss for samples with larger diameters. Weight losses for the second decay class (G2) were different, with smaller diameter samples showing greater length in the DTG curve (close to 295 °C). DTG peaks ranged from 348-361 °C, with larger diameters having higher peaks. The third decay class (G3) presented a wide range of weight losses, as well as in G2. The first DTG peak (close to 297 °C) did not show significant differences for the diameter classes of the CWDs. However, the maximum decomposition temperature of the second peak (close to 358-366 °C) increased as the CWD diameter increased. The last decay class (G4) showed a lower weight loss compared to the other decay classes. The DTG peaks (close to 277 °C) had a longer length for samples with larger diameters.
Weight losses and residual mass were quantified for each diameter and decay class of CWDs, in different temperature ranges ( Table 5). The weight loss ranged from 0.00 to 1.55% in the first temperature range (100-200 °C), from 10.84 to 18.65% in the second temperature range (200-300 °C) and from 25.63 to 65.13% in the last temperature range (300-450 °C). The residual mass at 450 °C ranged from 21.54 to 60.59%, being higher in the more decomposed CWDs.
Statistical analysis. The Kruskal-Wallis test indicated that differences between weight loss medians in the first (100-200 °C) and second (200-300 °C) temperature ranges, by decay class, were not significant (P > 0.05). On the other hand, ANOVA indicated a significant difference in the range of 300-450 °C and for residual mass at 450 °C (P < 0.05), being the class 4 decomposition mean, different from the other classes by Tukey test (Fig. 3). In this way, the thermogravimetric analysis was able to differentiate CWD samples into two groups, the first involving decay classes 1, 2 and 3 and the second group involving only decay class 4. The differences between the residual mass averages, by CWD's diameter classes, were not significant (P > 0.05).
In this way, the Spearman correlation was calculated for the weight losses in the temperature range of 300-450 °C and for the residual weight at 450 °C, separating the statistically different groups by the Tukey test into: Group 1 (G1)-decay classes 1, 2 and 3; Group 2 (G2)-decay class 4. The physical and chemical properties Table 4. Volatiles-Vol (%) (mean ± standard deviation), Ash content-Ash (%) (mean ± standard deviation) and Fixed Carbon-C (%) (mean) by diameter and decay classes of CWDs. Decay classes: (i) Materials that have just fallen to the ground with leaves and bark intact; (ii) Materials similar to those of class "i", but with the bark showing rotting or peeling; (iii) Materials with a high stage of decomposition and showing some resistance to being broken; (iv) Materials that are rotten, friable and without resistance to being broken. Means followed by the same letter do not differ statistically by Tukey's test, at the 5% significance level. www.nature.com/scientificreports/  www.nature.com/scientificreports/ used to calculate the correlation were: volatile materials (%), ash content (%), fixed carbon (%), C/N ratio and apparent density (g cm −3 ). The weight loss of CWDs in the temperature range of 300-450 °C of G1 showed a positive and stronger correlation with volatile materials, C/N ratio and density. The weight loss of G2 was positively associated only with volatile materials. The residual weight at 450 °C of G1 showed a positive correlation with ash and fixed carbon content, while G2 was also positively correlated with these variables plus the C/N ratio (Fig. 4).

Discussion
Physical and chemical properties of CWDs. The decomposition of CWDs in forest ecosystems is a crucial pathway for nutrients return to soil 59 . During this process, CWDs on the forest floor undergo different transformations in their chemical and physical properties, such as a reduction in density, an increase in water content, accumulation of nutrients and lignin, and a reduction in pH 60 . Most of these physicochemical transformations were observed in this study.
The results indicated a reduction of the average apparent density in function of the increase of CWDs decomposition (Table 2). This reduction in density can be explained by the weight loss due to wood-decomposing microorganisms' action 61 . Previous studies have also found this pattern of data behavior for CWDs density [37][38][39]50 .
The decomposition process had less impact on Carbon (C) concentrations, which did not show great variations with the decay class of CWDs increase ( Table 3). The higher concentration of lignin in CWDs may be one of the factors limiting the C degradation, since it's a large and complex structure, thus, difficult to decompose 37,[62][63][64] . In addition, this wood constituent can also compromise the degradation of cellulose and hemicellulose when incorporated in large amounts into cell wall structures 26 .
Nitrogen (N) contents also showed low variation with increasing decay classes of CWDs (Table 3). However, this behavior was not expected since this nutrient tends to accumulate over the years due to the fixation and Table 5. Weight loss (%) as a function of temperature range and residual mass at 450 °C by diameter and decay classes of CWDs. Decay classes: (i) Materials that have just fallen to the ground with leaves and bark intact; (ii) Materials similar to those of class "i", but with the bark showing rotting or peeling; (iii) Materials with a high stage of decomposition and showing some resistance to being broken; (iv) Materials that are rotten, friable and without resistance to being broken.

Decay classes Diameter classes (cm)
Weight loss (%) www.nature.com/scientificreports/ translocation of this chemical element from the soil to CWDs by heterotrophic microorganisms 1,65-67 . Besides, the increase in structural bonds between nitrogen and elements more resistant to degradation such as lignin, aromatic and phenolic compounds may also favor N accumulation during the wood decomposition process 68 . The C and N contents found in our study resulted in lower C/N ratio values for the more decomposed CWDs (Table 3). This pattern of declining C/N ratio was also found in studies conducted in tropical and subtropical forests 37,38,69,70 . A low C/N ratio of CWDs indicates a greater potential for wood decomposition causing these materials to remain a shorter time in the forest ecosystem 71,72 .
Immediate chemical analysis. The results indicated a tendency of volatile materials reduction with the CWDs decomposition (Table 4). This reduction in volatile materials may be correlated with the degradation of holocellulose and the decrease in extractive contents 73 . During the wood decomposition process, holocelluloses are readily degraded by decomposing microorganisms due to the greater ease of breaking down their structures compared to other compounds, such as lignin 74 . In the case of extractive contents, the reduction in their concentration occurs due to processes such as enzymatic deactivation, auto-oxidation, microbial degradation or by leaching 1 .
The ash contents showed a contrary trend to the volatiles, increasing their concentration with CWDs decay class increase (Table 4). These results suggest that CWDs accumulate inorganic nutrients such as potassium, calcium, magnesium and silicon in their composition as they lose weight and carbon through the decomposition process 38,66,75 . In addition, contamination by soil residues may also contribute to the higher ash content in the most decomposed CWDs. However, there is no way to measure the impacts of this contamination on our results.
Fixed carbon was not related to CWDs decay class in this study ( Table 4). The fact that this component of immediate chemical analysis is obtained by difference may have influenced these results. However, fixed carbon presented values inversely proportional to volatile materials, indicating a negative correlation with holocelluloses and a positive correlation with lignin 37,68 . Thermogravimetric analysis. Thermogravimetric analyzes indicated that samples with a high degree of decomposition have high residual mass and low weight loss. This pattern can be explained by the greater loss of carbon in the forest ecosystem due to the respiration of microorganisms, photodegradation and leaching 76,77 . Thermogravimetric analysis also indicated that hemicellulose decomposition occurs at lower temperatures (225-275 °C). The decomposition of hemicellulose in this temperature range is related to its amorphous chemical structure 78 , composed of sugars such as pentoses and hexoses 79,80 . Thermal degradation of cellulose occurs at 275-375 °C due to its crystallinity 81 . Lignin generally begins to decompose at lower temperatures 82 , but it is the last wood compound to fully decompose due to its cross-linked structure and high molecular weight 79 .
Furthermore, TG/DTG curves indicated different behaviors for the CWDs in the different size and decay classes. The longer lengths of the DTG curve, such observed in the first decay classes (G1), indicate higher concentrations of holocelluloses in samples with larger diameters. In addition, the lower residual weight for samples with larger diameters demonstrate that the rate of decomposition in the forest ecosystem is higher for www.nature.com/scientificreports/ smaller CWDs 77 . The distinct weight losses, such observed in the second (G2) and third (G3) decay classes, can be explained by the wide variety of tree species present in the forest. Less weight loss and greater residual weight, as observed in the last decomposition class (G4), demonstrate that the structural components of wood have already been significantly degraded in the forest ecosystem. Comparing the four decay classes, the DTG peaks were shifted to higher temperatures with increasing decay class, which is related to the removal of extractives. In fact, as the decomposition process occurs, the extractives are released and the temperature of thermal degradation of cellulose and hemicellulose shifts to higher temperatures 83 . Analysis of variance and Tukey's test indicated statistical difference only for the averages of weight loss in the last temperature range (300-450 °C) and for the averages of residual weight at 450 °C, differing between the decay classes 1, 2 and 3 (Group 1) of decay class 4 (Group 2) (Fig. 3). The difference between Groups 1 and 2 can be explained mainly by the apparent density and the C/N ratio, which are highly impacted variables throughout the CWD decomposition process (Fig. 4). In this way, the determination of physical and chemical properties, together with immediate and thermogravimetric analysis, can be considered an important tool to study the effects of CWDs decomposition process, defining with greater precision the classes of CWDs decomposition.
Our results narrowed the existing uncertainties in understanding the natural decomposition dynamics of CWDs. Although our study area is limited, the new guidelines used to determine the physical-chemical www.nature.com/scientificreports/ parameters of CWDs will serve as a basis for the sustainable management of this component and for the refinement of international reports that aim to quantify the carbon balance in forest ecosystems. Future research should focus on performing these analyzes at the species level, since this factor has a great influence on the physicochemical properties of CWDs 38,39 . Furthermore, it is recommended to carry out the microbiota characterization present in the soil and in the CWDs to distinguish the types of microorganisms and their abilities to degrade each constituent of the wood 1,84 . Finally, climatic factors such as temperature and humidity must also be considered as they influence the activity and selection of these microorganisms in the forest ecosystem 27,85 .

Conclusions
The apparent density of CWDs is affected by the natural process of decomposition while the carbon and nitrogen contents are less impacted by this process. The C/N ratio decreased directly with the decomposition class of the CWDs. The size classes of CWDs are not relevant for determining these properties. The physical-chemical properties must be always quantified according to the decay classes of the CWDs due to the diversity of species and the climatic conditions of each forest ecosystem. The CWDs structural chemical composition is affected by decomposition, resulting in loss of holocelluloses and extractives and an increase in lignin and ash concentration throughout this process. The weight loss is greater for the less decomposed CWDs and the residual weight is greater for the more decomposed ones. The smallest diameter classes have less weight loss and greater residual weight. The use of immediate chemical and thermogravimetric analysis removes the subjectivity to classify CWDs decomposition stages, reducing the number of tests to determine the physical and chemical properties of CWDs.
Our results contribute to a better understanding of the decomposition dynamics of CWDs and provide important information about their ecological role. The technical guidelines presented in this study should be applied and improved in other forest ecosystems around the world to increase the accuracy of scientific studies and international reports focused on the carbon cycle of these materials.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/